Fibrinogen and thrombin are critical proteins involved in achieving hemostasis after vascular injury and essential to blood clot formation. Fibrinogen and thrombin can be combined in powder form or in a non-aqueous suspension, without initiating a typical clotting reaction, thus preventing the formation of a fibrin clot until the proteins are hydrated in an aqueous medium or other liquid environment in which the proteins are soluble. An admixture of these proteins in powder form have a variety of potential biomedical applications including topical hemostasis, tissue repair, drug delivery, etc. In addition, an admixture of these proteins may be loaded onto a carrier or substrate, or other medical device, in powder form to form a product that may be used for example as a hemostatic device.
The clotting activity of thrombin is usually measured by combining thrombin in solution with a known amount of fibrinogen in solution. With appropriate conditions, the rate of clot formation after combining the two proteins is dependent on the activity of the thrombin. The rate of clot formation of a sample with an unknown amount of thrombin is compared with the rate of clot formation of a thrombin reference or thrombin standard to determine the activity of the sample.
Thrombin activity is a critical attribute of any thrombin/fibrinogen product and will dictate its functionality. Although the measurement of free thrombin is straightforward, measurement of thrombin activity when thrombin and fibrinogen are in an unreacted admixture has been a challenge since measurement thereof typically requires that the admixture of proteins be hydrated and solubilized, and fibrin clot formation between solubilized thrombin and fibrinogen immediately initiates upon hydration. Furthermore, since thrombin is known to bind and interact specifically with the immediately formed fibrin clot, the thrombin becomes bound in the fibrin clot and is no longer freely soluble in the hydrating solution and becomes unavailable for subsequent measurement of thrombin activity. Hence, any resultant measurement of the thrombin activity of any thrombin/fibrinogen product via hydration and clot formation is only partial and hence inaccurate.
Moreover, when the proteins are in an unreacted admixture and loaded onto a carrier, substrate or medical device, it may be necessary to remove the proteins from the substrate to accurately measure the thrombin activity, for example, if the carrier, substrate or medical device adversely affects the measurement of the activity or functionality of the proteins due to physical, chemical or optical interference with the measurement detection system. To overcome interference from the carrier, substrate or medical device, removal or extraction of the proteins must be performed without exposing the admixture to aqueous conditions, which would result in clot formation preventing subsequent measurement.
Fibrinogen is most frequently measured by a method originally described by Clauss, which measures fibrinogen functionality based on the rate of clot formation. In a typical Clauss assay, a sample with an unknown amount of soluble fibrinogen is combined with an excess of thrombin. The proportions of fibrinogen and thrombin are such that fibrinogen is the rate limiting reactant and the rate of clot formation is a function of the fibrinogen concentration. A fast clotting time would be indicative of a high fibrinogen concentration. Conversely, a longer clotting time would indicate a low concentration of functional fibrinogen. The amount of functional fibrinogen can be quantified by comparing the clotting time of the sample with those of a series for standards to establish a standard curve. The concentration of fibrinogen in the sample can be determined mathematically based on the equation derived from the clotting times of the standards.
While measurement of free fibrinogen in solution, e.g., human plasma, can be performed by established methods, assessment of fibrinogen functionality when fibrinogen is in the presence of thrombin has been a challenge. Hydration of the admixture will result in thrombin-mediated conversion of fibrinogen into an insoluble fibrin clot. Once fibrin is generated, any subsequent measurement of fibrinogen is no longer possible since the release of the fibrinopeptides from fibrinogen resulting in fibrin formation is essentially irreversible.
Hence there remains a need to accurately measure the activity of thrombin in the presence of fibrinogen, and to measure the functionality of fibrinogen in the presence of thrombin.